Enhancing monolignol ferulate conjugate levels in poplar lignin via OsFMT1

Background The phenolic polymer lignin is one of the primary chemical constituents of the plant secondary cell wall. Due to the inherent plasticity of lignin biosynthesis, several phenolic monomers have been shown to be incorporated into the polymer, as long as the monomer can undergo radicalization so it can participate in coupling reactions. In this study, we significantly enhance the level of incorporation of monolignol ferulate conjugates into the lignin polymer to improve the digestibility of lignocellulosic biomass. Results Overexpression of a rice Feruloyl-CoA Monolignol Transferase (FMT), OsFMT1, in hybrid poplar (Populus alba x grandidentata) produced transgenic trees clearly displaying increased cell wall-bound ester-linked ferulate, p-hydroxybenzoate, and p-coumarate, all of which are in the lignin cell wall fraction, as shown by NMR and DFRC. We also demonstrate the use of a novel UV–Vis spectroscopic technique to rapidly screen plants for the presence of both ferulate and p-hydroxybenzoate esters. Lastly we show, via saccharification assays, that the OsFMT1 transgenic p oplars have significantly improved processing efficiency compared to wild-type and Angelica sinensis-FMT-expressing poplars. Conclusions The findings demonstrate that OsFMT1 has a broad substrate specificity and a higher catalytic efficiency compared to the previously published FMT from Angelica sinensis (AsFMT). Importantly, enhanced wood processability makes OsFMT1 a promising gene to optimize the composition of lignocellulosic biomass. Supplementary Information The online version contains supplementary material available at 10.1186/s13068-024-02544-y.

trees were then moved to two-gallon pots containing perennial soil (50% peat, 25% fine bark and 25% pumice; pH 6.0), in a greenhouse where they were maintained on flood tables with supplemental lighting (16-h days) and watered daily with fertilized water.
The trees were harvested, the leaves and bark were removed, and the developing xylem was scraped from the stems.Xylem scrapings, bark and leaf tissue were stored at −80 °C, while the remaining stem was left to air dry.
RNA was isolated from developing xylem tissue using the CTAB-based method described by (Kolosova et al., 2004).Contaminating DNA was removed using a DNase I DIGEST kit (Ambion, ThermoFisher Scientific), and 1 µg of DNase-treated RNA was used to generate cDNA with the iScript cDNA synthesis kit (Bio-Rad Labs).The resulting cDNA was stored at -20 °C until use.Realtime quantitative PCR (RT-qPCR) reactions consisted of 10 µL of SsoFast Eva Green Supermix (Bio-Rad Labs), 20 pmol of primers, 1 µL of cDNA, and deionized water to a total volume of 20 µL.qPCR was performed using a CFX 96 System (Bio-Rad Labs) with the following primers: OsFMT RT FW2 5´-TTGGAGCAGCACAATTCATC-3´ and OsFMT RT RV2 5´-AAAGGACTGGAACGATGGTG-3´.For the housekeeping gene, TIF was used, with the following primers: TIF FW: 5´-CTGATAACACAAGTTCCCTGC-3´, and TIF RV: 5´-GACGGTATTTTAGCTATGGAATTG-3´.The following thermal cycler regime was used to amplify the 143-bp fragment: 30 s at 95 °C, 39 cycles of 95 °C for 5 s, and 57 °C for 30 s, followed by 95 °C for 30 s, and a melt curve cycle of 55 °C to 95 °C with an increment of 0.5 °C for 5 s.

Autofluorescence microscopy
Petioles from mature leaves of WT and transgenic poplar plants were harvested and persevered in 50% ethanol under refrigeration.Petioles were rehydrated overnight in 100% deionized water and trimmed 2 cm above the stem attachment point.Sections of ~100 µm were prepared with a RapidTome device (Thomas et al., 2023), placed on a slide, treated with a drop of 0.1 M NH4OH, sealed with a cover slip, and their blue autofluorescence imaged with the 5x objective of a Leica DMI3000 B inverted fluorescence microscope equipped with a standard DAPI filter cube.Identical parameters were used for all images (exposure time 298.5 ms, gain 2.0, saturation 0.75, gamma 0.99).As cell wall ferulate is characteristic of commelinid monocots but typically absent from dicots (Harris and Trethewey, 2010), sections of a stem of the grass teosinte (Zea diploperennis), obtained from the WSU biology greenhouse diversity collection, served as a positive control, and a stem of a dicot, barberry (Berberis sp.), served as a negative control.

Supplemental Table
Figure S1.Expression of OsFMT in transformed poplars.The expression levels of OsFMT in xylem tissue are relative to the housekeeping gene TIF5a.No expression of OsFMT was observed in WT trees.n = 3 biological replicates for each line (each with two or three technical replicates).Error bars represent standard error of the mean (SEM).Supplemental Figure S2.Height and diameter of OsFMT transgenic poplars.A) Diameter (mm) and B) Height (cm) of OsFMT transformed poplars and WT poplars, measured following 5 months of growth in a greenhouse.n = 7 or 8 biological replicates for each line.Error bars represent SEM.Statistical differences were determined via ANOVA and Dunnett's post-hoc test: **0.01 > P > 0.001.Supplemental Figure S3.Autofluorescence microscopy of petiole cross-section of OsFMT poplars and controls.Petioles sections were treated with either water (No Base) or 0.1 M ammonium hydroxide (With Base).Ammonium hydroxide treatment should intensify phenolic acids' autofluorescent signal and that from their ester derivatives.Petiole cross-sections of poplar plants transgenic for OsFMT (denoted by numbers) showed greater cell wall autofluorescence with a DAPI filter set than wild-type (WT) plants.The monocot Zea diploperennis (Zd) is shown as a positive control, and a dicot Berberis sp.(B.sp.) as a negative control.Magnification 50x, scale bar is 1 mm.Supplemental Figure S4.Saccharification efficiency of OsFMT, AsFMT, and WT poplar trees.A) Released glucose at the 4 h timepoint.B) Released glucose at the 24 h timepoint.C) Released glucose at the 48 h timepoint.D) Released xylose at the 4 h timepoint.E) Released xylose at the 24 h timepoint Released xylose.F) Released xylose at the 48 h timepoint.n = 3 biological replicates for each line (each with two technical replicates).AsFMT = AsFMT line 7, the highestexpressing line from Wilkerson et al. 2014.Error bars represent SEM.Statistical differences were determined via ANOVA and Dunnett's post-hoc test: *0.05 > P > 0.01; **0.01 > P > 0.001; and ***P < 0.001.

Table S2 .
The quantified DFRC monomers reported for the native lignin monomer and not that of the derivatized substructures.Values are the average of n = 3 biological replicates run in duplicate, with the standard error of the mean (SEM).ND = not detected.Bold values correspond to a statistical difference, determined via ANOVA and Dunnett's post hoc test, P < 0.05.No significant differences were calculated for SpCA and SFA, as these were not detected in WT.

Table S3 .
UV-Vis spectra of preparations of EL (enzyme lignin) for estimations of ferulate content.Values are the average of n = 3 biological replicates run in duplicate, with the standard error of the mean (SEM).